Enzymes are widely used in the food and feed industries such as in the fruit and vegetable processing industry.
In recent years, using enzymes to process fruit (including berry) mash has become state-of-the-art in juice e.g. citrus, apple and pear production as highest possible yields and throughput have become paramount. Pectin, a natural substance found in all fruit acts as cell glue, giving structure to the fruit. In fruit mashes, pectin binds with water to increase the viscosity and makes it difficult to release the juice in the liquid/solid separation by presses or decanters. The use of enzymes allows producers to control production according to demand.
In citrus fruit processing, the application of enzymes reduces juice viscosity, eliminating the risk of jellification during fruit juice concentration and storage and securing high cloud stability. Applied during the pulp extraction process, enzymes facilitate the release of juice and solids, increasing yield and improving cost efficiency. A stable cloud—or, in the case of lemon juice, successful clarification—is an important measure of the final product's superior quality. Essential citrus oils can also be recovered while using enzymes in citrus fruit processing. Apart from providing access to a valuable commodity, citrus oil recovery is important from a sustainability point of view, ensuring a low content of the bactericidal oils in wastewater for improved wastewater biodegradability and reducing overall water consumption. Enzymes are also used in the production of high clarity apple and pear juice which is dependent on the complete breakdown of starch and pectin content. This has been achieved by the addition of pectinases and starch-degrading enzymes to the juice before clarification and filtration. Added to the fruit mash before pressing and juice separation, pectinases lead to a reduction in mash viscosity, resulting in increased juice yield and optimised processing capacity. Less waste pomace and easier cleaning of fruit presses are other value-adding advantages. Also in wine production enzymes are used for transferring valuable components, such as aromas, colour and tannins, from the grape to the wine. Enzymes can furthermore help to preserve wine quality, sometimes over many years in storage. They also reduce fermentation time and promote clarification, filtration and stabilisation.
As mentioned above enzyme preparations are often used during fruit juice manufacture in the steps of extraction and liquefaction of fruit and fruit juice clarification. The commercial enzyme preparations contain a mixture of mainly pectinases (e.g. polygalacturonases, pectin esterases, pectin lyases) together with minor quantities of other hydrolytic enzymes such as arabinanases, galactanases and xylanases. The substrates for the various pectinases are pectins, which are polygalacturonides of high molecular weight (20-100 kDalton) consisting of alpha-1,4-glycosidic bound D-galacturonic acid polymers. Some of the uronic acid groups are esterified with methanol. The polygalacturonic backbone is interrupted by so-called hairy regions, consisting of a rhamnose-galacturonic acid backbone with arabinose-rich side chains.
The pectin degrading enzymes like polygalacturonase, pectin methyl esterase, pectin lyase or pectate lyase are important, especially in fruit and vegetable processing such as fruit juice processing or wine making, where their ability to catalyse the degradation of the backbone of the pectin polymer is utilized.
Pectins occur in nature as constituents of higher plant cell walls. They are found in the primary cell wall and middle lamella where they are embedded in cellulose fibrils. The composition of pectin and the degree of methylation is variable among plant species and moreover dependent on the age and maturity of the fruit. Among the richest sources of pectins are lemon and orange rind, which can contain up to 30% of this polysaccharide. Pectinases can degrade the carbohydrate polymer either by hydrolysis of the alpha-1,4-glycosidic bond (endo and exopolygalacturonases) or by transelimination reaction (pectin lyases). Pectin esterases can demethylate highly esterified pectin into polygalacturonic acid. Pectin lyases are specific for highly esterified pectins, polygalacturonases hydrolyse low esterified pectins. Consequently highly esterified pectins can be degraded by pectin lyases or the combination of pectin esterases and polygalacturonases.
In the various stages of fruit and vegetable processing pectinases play an important role. Originally pectinases were used for treatment of soft fruit to ensure high yields of juice and pigments upon pressing and to clarify raw press juices. Polygalacturonases are used as macerating enzymes for the production of pulpy nectars, loose cell suspensions that are the result of limited pectin breakdown particularly in the middle lamella. A combination of several pectinases together with cellulolytic enzymes is needed to almost completely liquefy fruit tissue, thereby facilitating extraction. The clarification of apple juices can for example be improved by the combined activity of pectin esterases and polygalacturonases or by pectin lyases for which the highly esterified apple pectin is an ideal substrate.
Most of the pectinases present in commercial preparations are of fungal origin. Aspergillus niger (A. niger) is a very important organism for the industrial production of pectin degrading enzymes. In A. niger the various pectinases are not expressed constitutively. Pectin or degradation products of the pectin molecule are needed as inducing substances. The fermentation conditions for pectinase production often result in a wide spectrum of pectinases. Moreover, A. niger produces many isoenzymes of the various pectinases. Several patents have published describing that genes encoding polygalacturonase (EP 421 919, EP 0 388 593), pectin lyases (EP 0 278 355, EP 0 353 188) and pectin esterases (EP 0 388 593) have been isolated and used for the construction of overproducing transformants. These transformants allow the production of specific enzymes, needed e.g. in maceration applications and in studies on the effect of the various pectinases in processes like liquefaction and clarification.
The isolation and characterization of a cell-wall polysaccharide from apple juice obtained after the liquefaction process in which the juice was released from the apple pulp by the combined action of pectolytic and cellulolytic enzymes has also been described. These cell-wall polysaccharides resemble the hairy regions of apple pectin (a rhamnose-galacturonic acid backbone with arabinose rich side chains) and have been called Modified Hairy Regions (MHR). Hairy regions are known to be present not only in apples but also in carrots, grapes and strawberries and are probably a common part of pectin molecules. The modified hairy regions are resistant to breakdown by the enzymes present in most pure and technical pectinase and cellulase preparations. A commercial crude enzyme preparation obtained from Aspergillus aculeatus has been found to be able to depolymerize the rhamnogalacturon backbone of these fragments. This activity was made visible by measuring the shift in molecular weight distribution using High Performance Gel Permeation Chromatography (HPGPC). The enzyme responsible for the degradation of the modified hairy regions prepared from apple juice are called rhamnogalacturonase (RGase). The enzyme can split glycosidic linkages in the rhamnogalacturonan backbone of (apple) pectins producing, besides other not yet fully identified reaction products, a range of oligomers composed of galacturonic acid, rhamnose and galactose with rhamnose at the non reducing end, hence the name rhamnogalacturonase (RG-ase) for this novel enzyme.
U.S. Pat. No. 5,591,620 describe the isolation of an Aspergillus gene encoding rhamnogalacturonase (RG-ase) and the construction of recombinant Aspergillus strains with overexpression of RG-ase. These strains can be used for the commercial production of RG-ase.
EP 1658359 describes a preparation comprising a mass consisting of olives or olive constituents and an enzymatic mixture containing at least one pectinesterase, at least one endopolygalacturonase and at least one exopolygalacturonase. In said preparation the ratio of the pectinesterase activity to the endopolygalacturonase activity is at least 0.13 and the ratio of the pectinesterase activity to the exopolygalacturonase activity is at least 0.3.
WO2008/117301 relates to a process for the production of a multienzyme system by fermentation. WO2008/117301 also describes the use of Aspergillus oryzae MTCC 5154 under optimized conditions for the production of a multienzyme system by submerged fermentation. The product described is a mixture of different enzymes such as α-amylase, amyloglucosidase, carboxy methyl cellulase, pectinase, beta-galactosidase and xylanase.
In “Aspergillus Enzymes Involved in Degradation of Plant Cell Wall Polysaccharies”, Microbiology and Molecular Biology Reviews, December 2001, p. 497-522 de Vries et al describes different classes of enzymes involved in plant cell wall polysaccharide degradation produced by Aspergilli, the genes encoding these enzymes, and the regulation of these genes. The cell wall degrading activities that have been detected from Aspergillus strains are furthermore described.
WO097/43423 describes purification of an α-glucuronidase from a crude, mixed enzyme preparation Pektinase 146, which is marketed as a pectinase source derived from an Aspergillus strain having the reference number 4M146.
Commercial preparations containing a number of different degrading enzymes are also available.
However, as highest possible yields and throughput are increasingly important in industrial processes more efficient processes providing enzymatic solutions are still needed.